The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including otitis media, pneumonia and meningitis. Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with S. pneumoniae, many questions concerning the virulence of this microbe remain.
While certain Streptococcal factors associated with pathogenicity have been identified, e.g., capsule polysaccharides, peptidoglycans, pneumolysins, PspA Complement factor H binding component, autolysin, neuraminidase, peptide permeases, hydrogen peroxide, IgAl protease, the list is certainly not complete. Further very little is known concerning the temporal expression of such genes during infection and disease progression in a mammalian host. Discovering the sets of genes the bacterium is likely to be expressing at the different stages of infection, particularly when an infection is established, provides critical information for the screening and characterization of novel antibacterials which can interrupt pathogenesis. In addition to providing a fuller understanding of known proteins, such an approach will identify previously unrecognized targets.
The enzyme UDP-N-acetylglucosamine enolpyruvyltransferase, encoded by the gene MurA (MurZ), catalyses the first committed step of peptidoglycan biosynthesis. The gene has been cloned and sequenced from Escherichia coli and the corresponding enzyme (419 amino acids) has been over-expressed and purified (Marquardt, J. L. Siegele, D. A., Kolter, R. & Walsh, C. T. (1992) J. Bacteriol. 174, 5748-5752). The kinetic mechanism of this enzyme has been well characterized. Enolpyruvate is transferred from phosphoenolypyruvate (PEP) to UDP-N-acetylglucosamine (UDPGlcNAc) with the release of inorganic phosphate. A cysteine residue (115) at the active site of the enzyme has been shown to be important in the enzyme mechanism and is the residue to which phosphomycin, an irreversible inhibitor of the enzyme, binds (Wanke, C. & Amrhein, N., (1993) Eur J. Biochem. 218 861-870; Marquardt, J. L., Brown, E. D., Lane, W. S., Haley, T. M., Ichikawa, Y., Wong, C-H., Walsh, C. T. (1994) Biochemistry 33 10646-10651.; Kim, D. H., Lees, W. J., Kempsell, K. E., Lane, W. S., Duncan, K. & Walsh, C. T. (1996) Biochemistry 35 4923-4928. ) The MurA gene has been shown to be essential in E. coli (Brown, E. D., Vivas, E. I., Walsh, C. T. & Kolter, R. (1995) J. Bacteriol. 177, 4194-4197.) and has also been cloned from Bacillus subtilis, Enterobacter cloacae (Wanke, C. Falchetto, R. & amrhein, N. (1992) FEBS Lett 301, 271-276), Mycobacterium tuberculosis and Acinetobacter calcoaceticus (Ehrt, S. & Hillen, W. (1994) FEMS Microbiol. Lett, 117, 137-142). The discovery of a MurA homologue in the human pathogen Streptococcus pneumoniae allows production of UDP-N-acetylglucosamine enolpyruvyl transferase enzyme which can then be used to screen for novel antibiotics as described below. Inhibitors of this protein have utility in antibacterial therapy as they will prevent the construction of the bacterial cell wall.
Clearly, there is a need for factors that may be used to screen compounds for antibiotic activity and which may also be used to determine their roles in pathogenesis of infection, dysfunction and disease. There is a need, therefore, for identification and characterization of such factors which can play a role in preventing, ameliorating or correction infections, dysfunctions or diseases.
The polypeptide of the present invention has amino acid sequence homology to known UDP-N-acetylglucosamine enolpyruvyl transferase. The amino acid sequence of SEQ ID NO:2 is the translated open reading frame sequence of SEQ ID NO:1 and displays homology (51% identity) to the UDP-N-acetylglucosamine enolpyruvyl transferase from E. coli.